Passive electric field focus system for in vivo and in vitro applications

ABSTRACT

The present invention provides a system and method of directing, focusing, or concentrating electrical charges within a defined electric field so that these charges can be used to exert forces on cells and tissues in vivo and/or cell cultures in vitro. The present invention reduces and/or eliminates the damage at a target site that would normally be caused by an electrode that acts as a current source or sink to accomplish the same task.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior filed InternationalApplication, Ser. No. PCT/US2007/008662 filed Apr. 9, 2007, which claimspriority to U.S. provisional patent application No. 60/744,424 filedApr. 7, 2006 and U.S. provisional patent application No. 60/745,530filed Apr. 25, 2006 which is hereby incorporated by reference into thisdisclosure.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.DGE-0221681 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Electroporation is known in the art as a method of treating a tissue inorder to transiently increase the tissue's permeability to enhancemolecular transport either for drug delivery or for sampling ofinterstitial fluids. Electroporation, or electropermeabilization,ellicits a significant increase in the electrical conductivity andpermeability of the cell plasma membrane caused by an externally appliedelectrical field. Electroporation and electropermeabilization are knownin the art. Some chemotherapeutic drugs used in cancer therapy have pooraccess into the tumor cells. Therefore, electropermeabilization offersan approach for increased drug delivery into the cells and thus betterantitumor effectiveness. Tissues can also be electropermeabilized andthus the antitumor effectiveness of chemotherapeutic drugs potentiatedby increasing drug delivery into the cells. It is known thatelectroporation can be performed both in vivo and in vitro.

Forces resulting from applied electric fields can be used on biologicalentities such as cells and tissues for many purposes that include cellbreakdown, electrophoretic extraction or insertion of molecules,including electroporation or electropermeability, in addition to otherforms of molecular manipulation know in the art.

In a system to apply electric fields to biological entities, a set ofelectrodes are used to establish an electric field within a biologicalentity. Currently, there is no system to direct, or focus, theestablished electric field intensity in in vivo and in vitro situationsto a specific location within an electric field defined by the set ofelectrodes.

SUMMARY OF INVENTION

The present invention consists of a system and method of focusingelectric fields for the purpose of exerting electrical forces onbiological entities.

In accordance with the present invention, a system and method areprovided for focusing an electric field intensity within a definedelectric field to exert electrical forces on a biological entity. In aparticular embodiment, the system includes, at least two activeelectrodes to establish an electric field within the biological entityand at least one passive electrode positioned within the path of theelectric field to focus the electric field intensity of the establishedelectric field at a desired location within the biological entity.

The system further includes a power source coupled to the at least twoactive electrodes to establish the electric field and software tocontrol the field attributes, such as duration and intensity level. Inthis configuration, one of the electrodes is considered the sourceelectrode and another of the electrodes is the sink electrode.

The system of the present invention is operation in both an in vivo andin vitro environment. In the case of an in vivo application, thebiological entity may be a cell or group of cells, or a cellular tissue.In the case of an in vitro application, the biological entity may be acell culture.

Various embodiments of the passive electrode are within the scope of thepresent invention. These embodiments include, but are not limited to, apassive electrode that is fabricated of a material from the groupincludes metals, semiconductors, nanoparticles, plastics, polymers,bio-polymers, fluidistic substances, bio-molecules, antibodies,proteins, DNA, RNA, non-conductive materials and high relativepermittivity materials.

Additionally, the geometric configuration of the passive electrode maytake on various forms. These forms include, but are not limited to, astar geometry, a four-corners geometry, a group of conductive needles,or a sphere for positioning below a surface level of the biologicalentity. The system of claim 1, wherein the passive electrode is shapedin a star geometry.

As such, the method of the present invention accomplishes the task offocusing the electric field within the biological entity by using apassive electrode(s) that does not act as a current source or sink forthe field as defined by the set of electrodes used to generate theelectric field. Accordingly, the present invention provides severaladvantages over existing technologies. The present invention provides ameans of directing electric fields for application to living cells andtissues, facilitates in vivo electroporation in tissues that are inlocations that are difficult to access, provides new applications andprotocols for existing proprietary electroporation and/or electrofusiontechnologies, reduces and/or eliminates direct harm to cells and tissuesat desired treatment sites caused by direct contact with activeelectrodes, and greatly improves the directional focus of the electricfield.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 is an illustrative example of an embodiment of the presentinvention wherein the electric field running between two electrodes isconcentrated in the perpendicular direction of the applied electricfield when using highly conductive materials.

FIG. 2 is an illustrative example of an embodiment of the presentinvention wherein electric fields are concentrated in the direction ofthe applied electric field when using materials with relatively highpermittivity.

FIG. 3 illustrates the results corresponding to the magnitude of theelectric field intensities resulting from the simulation wherein thereis no passive electrode.

FIG. 4 illustrates an embodiment of the present invention showing theresults corresponding to the magnitude of the electric field intensitiesresulting from a simulation wherein there is a passive electrode with a“star” geometry is positioned within the field.

FIG. 5 illustrates an embodiment of the present invention showing theresults corresponding to the magnitude of the electric field intensitiesresulting from a simulation wherein there is a passive electrode with a“four corners” applicator geometry.

FIG. 6 illustrates actual results for a prototype “star” geometryapplicator in accordance with the present invention.

FIGS. 7A and 7B illustrate the results of improved electroporation atthe site of an implantable device in accordance with the presentinvention.

FIGS. 8A and 8B illustrate the results of improved electroporation for aneedle electroporation application in accordance with the presentinvention.

FIG. 9 illustrates a simulation of an implantable electroporation devicethat concentrates electric fields under living tissue employing asphere.

FIG. 10 illustrates a simulation of an implantable electroporationdevice that concentrates electric fields under living tissue employing ahollow sphere.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The method in accordance with the present invention takes advantage of aset of electric phenomena that occurs with changes in materials throughan electric field's path. The electric field is redirected orconcentrated in different places depending on the materials anddifferent design geometries. How the method uses materials that vary ingeometries and electrical characteristics such as conductivity andpermittivity and different arrangements and geometries can be betterexplained by simple abstractions such as those in FIG. 1, where theelectric field running between two electrodes is concentrated in theperpendicular direction of the applied electric field when using highlyconducting materials, and in FIG. 2, where the electric field isconcentrated in the direction of the applied electric field when usingmaterials with relatively high permittivity.

With specific reference to FIG. 1, a 2D simulation illustrates anelectric field running from the left electrode 10 to the right electrode15. In this exemplary embodiment, the circles 20 in between theelectrodes are made of platinum with the background being a vacuum.Platinum is an exemplary material and other conductive materials arewithin the scope of the present invention. As illustrated with referenceto the accompanying legend 25, the highest electric field is shown asred shading and the lowest electric field is shown as blue shading. Assuch, the highly conductive circles 20 are effective in concentratingthe electric field in the vertical direction (y) 30 when the appliedelectric field is running in the horizontal direction (x) 35.

With specific reference to FIG. 2, a 2D simulation illustrates anelectric field running from the left electrode 10 to the right electrode15. In this exemplary embodiment, the circles 20 in between theelectrodes are made of mica with the background being a vacuum. Mica isan exemplary material and other materials having a substantially highpermittivity are within the scope of the present invention. Asillustrated with reference to the accompanying legend 25, the highestelectric field is shown as red shading and the lowest electric field isshown as blue shading. As such, the high relative permittivity of thecircles of mica 20 are effective in concentrating the electric field inthe horizontal direction (x) 35 when the applied electric field isrunning in the same horizontal direction (x) 35.

The system in accordance with the present invention consists of a set ofelectrodes, having at least one current source electrode and at leastone current sink electrode and a power source, a passive electrode andsupporting software. The set of electrodes and associated power sourceare used to establish an electric field either in vivo or in vitro. Thepassive electrode is then placed in close proximity to the establishedelectric field. The passive electrode is used to direct, concentrate,and/or focus charge at the location of the passive electrode and awayfrom the current source and sink electrodes that define the shape of theoverall electric field. This focused charge in an in vivo and/or invitro application dramatically reduces, if not eliminates, the adverseeffects of Joule heating and redox reactions that would occur at thelocation of the passive electrode if a current source or sink electrodewere used instead of the passive electrode.

In accordance with the present invention, a passive electrode refers toa device that does not contain a source of electrical energy.

With reference to FIG. 3, FIG. 4 and FIG. 5, an electrostatic simulationis illustrated as proof of concept of the invention. In this embodiment,a 5 cm plastic well with PBS solution is provided and four activeelectrodes 50 are used to apply current to the solution.

In FIG. 3, a passive electrode is not provided and the correspondingmagnitudes of the electric field intensities that result from thesimulation are illustrated, along with the legend 25 identify theassociated colors. As shown, the lowest field intensity value is drawnin dark blue 55 (starting at 0V/m), and the most intense is drawn in red60 (ending at 1.5V/m). It is expected that the areas where the electricfield intensity is highest are the areas where charge will be focusedand electroporation/electrofusion will be facilitated.

In FIG. 4, a passive electrode 65 with a “star” geometry is used and thecorresponding magnitudes of the electric field intensities that resultfrom the simulation are illustrated, along with the legend 25 identifythe associated colors. As shown, the lowest field intensity value isdrawn in dark blue 55 (starting at 0V/m), and the most intense is drawnin red 60 (ending at 1.5V/m). It is expected that the areas where theelectric field intensity is highest are the areas where charge will befocused and electroporation/electrofusion will be facilitated.

In FIG. 5, a passive electrode 70 with a “four corners” geometry is usedand the corresponding magnitudes of the electric field intensities thatresult from the simulation are illustrated, along with the legend 25identify the associated colors. As shown, the lowest field intensityvalue is drawn in dark blue 55 (starting at 0V/m), and the most intenseis drawn in red 60 (ending at 1.5V/m). It is expected that the areaswhere the electric field intensity is highest are the areas where chargewill be focused and electroporation/electrofusion will be facilitated.

The simulation results illustrated in FIG. 3-FIG. 6, are shown in a 2dimensional space, but applications and design of applications areaccomplished in a 3 dimensional space. The geometries presented hereserve to indicate concept feasibility. Actual applicator design will beadjusted to the reality of cell and/or tissue 3D space.

FIG. 6 illustrates how the concept of the present invention is proven bygenerating electroporation in the zone where the electric fields shouldbe higher according to the simulation in FIG. 4, as previouslydescribed. FIG. 6 illustrates actual results for a prototype “star”geometry passive electrode as described in the simulations presentedabove. The “greenish” color is the result of electroporation andsubsequent florescence of an electroporation sensitive biomarker. Thefour peripheral green circular areas in the figure indicate theelectroporation that has occurred at each of the four currentsource/current sink active electrodes. The color region in the middle ofthe figure reflects electroporation at the passive “star” configuredelectrode. The absence of color in the remaining area of the figureillustrates that electroporation is limited to the targetedelectroporation site at the location of the passive electrode.

The materials that compose the active electrodes and/or the passiveelectrodes include but are not limited to the following:

(1) Metals, (2) Semiconductors, (3) Nanoparticles, (4) Non-metals suchas plastic, Polymers, Bio-polymers, (5) Fluidistic substances, (6)Bio-molecules such as antibodies, proteins, DNA, and RNA, (7)Non-conductive materials and (8) High relative permittivity materials.

Additionally, the passive electrode can be anything that is in the pathof the electric field, and may be native to the biological system thatis being treated and thus does not need to be introduced prior to theelectric field being established. The passive electrode is positioned inthe path of the electric fields between the sink and the sourceelectrode(s). Passive electrodes can be inside or outside the biologicalentity, and are not limited to foreign entities, but in additional maybe intrinsic to the entity. For example, the active electrodes may bepositioned such that a bone within the biological entity becomes thepassive electrode.

The passive electrode may take the form of a fluid which can change itsshape, volume and conformation to change its field distributioncharacteristics. When the passive electrode is a fluid, the fluid willtake on different concentration profiles and 3D shapes over time. It isalso know that a fluid passive electrode can change its position or beinduced to change its position relative to the active electrodes asnecessary.

The passive electrode in accordance with the present invention may beinside or outside of the biological entity. In addition, it can remainpermanently with the biological entity or be removed after it has servedits purpose. Alternatively, the passive electrode may become an activeelectrode. For example, a passive electrode that experiences a chemicalreaction that results in a potential difference, or a passive electrodethat is grounded after it has served its purpose would result in achange from a passive electrode to an active electrode. Additionally,the electrical characteristics of the passive electrode may change sothat the electric field is affected differently. For example, thepassive electrode may be fabricated of a material that changesconductivity under temperature conditions.

When utilizing an in vivo application, the device and components couldbe located in any tissue of a living being not restricted to a singlegeographic localization.

Possible geometries are not limited to those in the proof of conceptdevice; such geometry was only used to demonstrate the principle behindthe application of electric fields by passive electroporation on livingcells and biomolecules.

With reference to FIGS. 7A and 7B, in an exemplary embodiment it isshown that improved electroporation can be accomplished at the site ofan implantable device. The concentration of electric fields can beinduced at a specific site by using micro-metallic beans or composedmaterial beads. In this embodiment an applied electric field isconcentrated in living tissue near the site of an implantable device invivo. In this embodiment, the electric fields are concentrated in thelarger green volume 85 in between the two electrodes 90 in the imagesbecause of micro-beads of conductive material that are suspended in ahydrogen or other type of biocompatible matrix.

With reference to FIGS. 8A and 8B, in an exemplary embodiment it isshown that improved electroporation can be accomplished utilizing aplurality of needles as the passive application. By using a combinationof materials as composing the needles, it is possible to direct currentsin specific directions or depths. FIGS. 8A and 8B show an electric fieldas it is applied from electrodes 95 located at the left and right in thevolume. In this embodiment, passive conductive needles 100 are added inbetween the active electrodes, with no other changes to geometries orvariables, the charges are concentrated at the location of the passiveneedles. This configuration improves current state of the art in vivoelectroporation devices.

With reference to FIG. 9 and FIG. 10, in an exemplary embodiment it isshown that improved electroporation can be accomplished utilizing animplantable electroporation device. FIG. 9 and FIG. 10 show a simulationof an implantable electroporation device 105 that concentrates electricfields under living tissue. The first configuration, illustrated in FIG.9, is an implanted sphere 105 that serves to concentrate chargesestablished by the electrodes 110 under living tissue conditions.Additionally, as illustrated in FIG. 10, the sphere can be either hollowor filled with a different material, and the surface of the sphere 105may have a set of shell particles 115 that help concentrate charges asshown by the higher electric fields (red dots around surface of sphere).Different configurations of this device can achieve many applications.Additionally the passive electrode may take the form of a container forany agent. For example, the passive electrode may be a hollow spherecontaining a drug for delivery to the biological entity.

In an additional embodiment, the passive electrode may function as ascaffold for biological structures. For example, the passive electrodemay function as a scaffold for bone growth when used in vivo.

In an additional embodiment, the passive electrode is labeled to allowthe identification of the position of the passive electrode within theentity at any point in time. The passive electrode may be fluorescentlabeled, radio-labeled, magnetically labeled or contrast agent labeled,in addition to other such labeling techniques known in the art.

It will be seen that the advantages set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense. It is also to be understood that the following claimsare intended to cover all of the generic and specific features of theinvention herein described, and all statements of the scope of theinvention which, as a matter of language, might be said to falltherebetween. Now that the invention has been described,

What is claimed is:
 1. A method of focusing an electric field within adefined electric field to facilitate electroporation within a biologicalentity, the method comprising the steps of: establishing an electricfield between at least one first active electrode and at least onesecond active electrode within the biological entity, the establishedelectric field having an electric field intensity concentration focus;and changing the focus of the electric field intensity concentration tofacilitate electroporation at a desired focus location within thebiological entity by positioning a passive electrode within the electricfield established between the at least one first active electrode andthe at least one second active electrode, wherein the passive electrodeis fabricated of a material selected from the group consisting ofmetals, semiconductors, nanoparticles, plastics, polymers, bio-polymers,fluidistic substances, bio-molecules, non-conductive materials and highrelative permittivity materials, wherein the passive electrode is not acurrent source or a current sink for the established electric field,wherein the passive electrode is never physically or electricallycoupled to a source of electrical energy and wherein the passiveelectrode is never physically or electrically coupled to another passiveelectrode or to a conducting element.
 2. The method of claim 1, whereinthe at least one first active electrode is a source electrode and the atleast one second active electrode is a sink electrode.
 3. The method ofclaim 1, wherein the establishment of the electric field is controlledby software means.
 4. The method of claim 1, wherein the biologicalentity is in vivo.
 5. The method of claim 1, wherein the biologicalentity is in vitro.
 6. The method of claim 1, wherein the passiveelectrode is a fluid.
 7. The method of claim 6, wherein a concentrationprofile of the fluid passive electrode changes over time.
 8. The methodof claim 6, wherein a shape of the fluid passive electrode changes overtime.
 9. The method of claim 1, wherein the passive electrode isinternal to die biological entity.
 10. The method of claim 1, whereinthe passive electrode is removable.
 11. The method of claim 1, whereinthe passive electrode is a container fbr an agent.
 12. The method ofclaim 11, wherein the agent is a drug for delivery into the biologicalentity.
 13. The method of claim 1, wherein the passive electrode is ascaffold for biological structures within the biological entity.
 14. Themethod of claim 1, wherein the passive electrode is labeled.
 15. Themethod of claim 1, wherein the passive electrode further comprises aplurality of electrical characteristics and said electricalcharacteristics vary dependent upon varying environmental conditions.16. The method of claim 15, wherein one of the plurality of electricalcharacteristics is conductivity, and said conductivity of the passiveelectrode changes under varying temperature conditions.